Intracellular concentration and distribution of the ubiquitous second messenger Ca.sup.2+ is tightly controlled by a number of pathways (Tsien et al, Ann. Rev. Cell Biol., 6:715-760 (1990)). The interaction of the pathways that mobilize and regulate free Ca.sup.2+ levels can result in highly complex and dynamic signaling patterns, such as Ca.sup.2+ oscillations and waves (Tsien et al, supra; Berridge et al, Curr. Opinion Cell Biol., 6:267-274 (1994); and Meyer et al, Ann. Rev. Biophys. Biophys. Chem., 20:153-174 (1991)). Pulsed perturbation of the concentrations of various second messengers, achieved by flash photolysis of caged inositol-1,4,5-trisphosphate, diacylglycerol, and Ca.sup.2+ (Walker et al, Nature(London), 327:249-252 (1987); Harootunian et al, Cell Calcium., 12:153-164 (1991a); Adams et al, J. Am. Chem. Soc., 110:3212-3220 (1988); Ellis-Davies et al, J. Org. Chem., 53:1966-1969 (1988); and Ellis-Davies et al, Proc. Natl. Acad. Sci., USA, 91:187-191 (1994)), has yielded highly specific mechanistic information about these dynamic phenomena (Harootunian et al, supra; and Harootunian et al, Science, 251:75-78 (1991b)). Although the role of second messengers themselves in dynamic signaling phenomena has been studied by photorelease techniques, the contribution of pathways that regulate second messenger levels remains unexplored.
The family of sarcoplasmic/endoplasmic reticulum Ca.sup.2+ ATPases (hereinafter "SERCA") that sequester Ca.sup.2+ into the sarcoplasmic reticulum (hereinafter "SR"), and endoplasmic reticulum (hereinafter "ER") are important regulators of cytosolic free Ca.sup.2+ levels (Lytton et al, J. Biol. Chem., 267:14483-14489 (1992)). It was postulated in the present invention that the effect of these pumps on Ca.sup.2+ oscillations and waves could be elucidated by the development of a method for the pulsed modulation of their activity. This could be accomplished by the preparation of a caged, reversible SERCA inhibitor.
SERCA inhibitors have been described by Thomas et al, In: A Practical Guide to the Study of Calcium in Living Cells (Meth. Cell Biol., 40), Academic Press, San Diego, pp 65-89 (1994). The three most commonly used SERCA inhibitors are thapsigargin (Thastrup et al, Proc. Natl. Acad. Sci., USA, 87:2466-2470 (1990); and Lytton et al, J. Biol. Chem., 266:17067-17071 (1991)), cyclopiazonic acid (Goeger et al, Biochem. Pharmacol., 38:3995-4003 (1989)) and DBHQ (Moore et al, FEBS Lett., 224:331-336 (1987); and Kass et al, J. Biol. Chem., 264:15192-15198 (1989)). Because its inhibitory action is irreversible (Sagara et al, J. Biol. Chem., 267:1286-1292 (1992)), thapsigargin is not a suitable target for a caged reagent to be used for reversible photomodulation of SERCA activity. Cyclopiazonic acid has a relatively complex molecular structure and, being a biosynthetic product of fungal origin, is available only in small quantities at high expense, which makes it an unattractive starting material for organic synthesis. In contrast, DBHQ is structurally simple, incorporating only one type of reactive functional group for caging purposes, and is commercially available in large quantities. These advantages, together with its reversibility, made DBHQ the preferred target for caging in the present invention.
The great majority of photoreleasable compounds have used caging groups structurally based on the 2-nitrobenzyl system (Kao et al, In: Optical Microscopy, Emerging Methods and Applications, Herman et al, eds., Academic Press, San Diego, pages 27-85 (1993)). Although the simple parent 2-nitrobenzyl moiety is a common caging group, it was not appropriate for caging DBHQ: preliminary experiments indicated that UV irradiation of cells bathed in medium containing 2-nitrobenzyl alcohol resulted in irreversible inhibition of the SERCA pump. Because photolysis of any 2-nitrobenzyl-caged compound is expected to generate the same photochemical byproducts, it was inferred in the present invention that the byproducts of photolyzing a 2-nitrobenzyl-caged DBHQ would not be inert.
It has been shown that the .alpha.-carboxy-nitrobenzyl (CNB) group is useful for caging neuroactive amino acids (Milburn et al, Biochem., 28:49-55 (1989); Gee et al, J. Am. Chem. Soc., 116:8366-8367 (1994); Wieboldt et al, Biochem., 33:1526-1533 (1994a); Wieboldt et al, Proc. Natl. Acad. Sci., USA, 91:8752-8756 (1994b); and Gee et al, J. Org. Chem., 60:4260-4263 (1995)). Photodeprotection was shown to proceed rapidly and with high quantum yield (Wieboldt et al, (1994b) supra). It was recognized in the present invention that the carboxylate on this caging group would reduce the reactivity of the photochemical byproduct. Furthermore, the presence of the carboxylate offers the added advantages of increasing the water solubility of the caged compound and allowing for the preparation of a caged AM ester, which could be passively loaded into cells.
Caging DBHQ directly with the CNB group requires the formation of a benzyl ether, which model studies indicated was problematic. For example, reaction of DBHQ with 2-nitrobenzyl chloride in the presence of K.sub.2 CO.sub.3 yielded numerous compounds that were difficult to isolate and characterize. Reasoning that the difficulties encountered in benzyl ether formation were at least partially the result of the sterically congested environment surrounding the phenolic hydroxyl groups of DBHQ, it was postulated in the present invention that an efficacious caging reaction would need to proceed through a different mechanism.
The o-nitromandelyloxycarbonyl (Nmoc) group was designed in the present invention as a photocleavable caging group that would combine the desirable qualities of the CNB group with a caging reaction that proceeds via carbonyl substitution. As shown in FIG. 1, irradiation of the caged-DBHQ with UV light would result in the formation of DBHQ-bicarbonate, which would rapidly decompose under physiological conditions to DBHQ and carbon dioxide. The photochemical side product, a 2-(2-nitrosophenyl)glyoxylate, is the same as that generated by photolysis of .alpha.-CNB-caged molecules (Milburn et al, supra; Gee et al, supra;
Wieboldt et al, (1994a) supra; Wieboldt et al (1994b), supra; and Gee et al, supra), for which no adverse biological effects have ever been reported. Carbon dioxide, liberated by decarboxylation of DBHQ-bicarbonate, is a normal product of metabolism, and would thus also be innocuous.
Thus, the present invention provides caged DBHQ. The compounds of the present invention make it possible to deliver, with temporal precision, controlled doses of DBHQ to spatially restricted sites in living biological samples.